Development of A Novel EvaGreen-Dye Based Recombinase Aided Amplification Assay Using Self-Avoiding Molecular Recognition System Primers

Abstract

Introduction: Fluorescent probe-based recombinase aided amplification (RAA) offers the advantages of rapidity and simplicity but is limited by the requirement for complex and lengthy probe design, restricting its widespread application.

Methods: A novel EvaGreen dye-based RAA (EvaGreen-RAA) assay utilizing self-avoiding molecular recognition system (SAMRS) primers was developed for the detection of Pseudomonas fluorescens (PF) and Bacillus cereus (BC) in milk. Conventional RAA was used as a reference method. Sensitivity was evaluated using nucleic acids from recombinant plasmids and simulated milk specimens. Additionally, a dual EvaGreen-RAA assay was investigated for simultaneous detection of mixed BC and PF in simulated milk specimens.

Results: The EvaGreen-RAA demonstrated superior sensitivity compared to conventional RAA, with detection limits of 1 copy/µL versus 10 copies/µL for both BC and PF plasmids, respectively. In simulated milk specimens, EvaGreen-RAA detected BC and PF at concentrations of 100 CFU/mL and 200 CFU/mL, respectively, compared to 400 CFU/mL and 600 CFU/mL for conventional RAA. The dual EvaGreen-RAA assay successfully detected mixed BC and PF in simulated milk specimens at concentrations of 200 CFU/mL for each pathogen.

Conclusion: The EvaGreen-RAA assay demonstrated significant advantages in terms of simplicity and enhanced sensitivity compared to fluorescent probe-based RAA, offering a novel approach for developing multiplex pathogen detection systems using melting curve analysis.

Keywords: Bacillus cereus; EvaGreen dye; Melt Curve; Pseudomonas fluorescens; Raw milk; Recombinase aided amplification; SAMRS.

Figure 1

Effect of SAMRS primers on primer dimerization. (A) Merged HRM results of the STD primer for BC; (B) Merged HRM results of SAMRS primers for BC; (C) Merged HRM results of the STD primer for PF; (D) Merged HRM results of SAMRS primers for PF; (E) AGE results of STD primers for BC; (F) AGE results of SAMRS primers for BC; (G) AGE results of STD primers for PF; (H) AGE results of SAMRS primers for PF. Note: Lane M: Marker; Lanes 1–6: 10¹, 10², 10³, 10⁴, 10⁵ and 10⁶ copies/μL BC or PF recombinant plasmids. Abbreviation: SAMRS=self-avoiding molecular recognition system; AGE=agarose gel electrophoresis; STD=standard nucleotide; HRM=high-resolution melting curves; NC=negative control; BC=Bacillus cereus; PF=Pseudomonas fluorescens.

Figure 2

Sensitivity assessment of RAA and EvaGreen-RAA on recombinant plasmids. (A) The sensitivity of RAA for BC was 10 copies/μL; (B) The detection sensitivities of RAA for PF was 10 copies/μL; (C) The sensitivity of EvaGreen-RAA for BC was 1 copy/μL; (D) The detection sensitivities of EvaGreen-RAA for PF was 1 copy/μL. Detection of RAA and EvaGreen-RAA on simulated milk specimens; (E) The detection sensitivity of RAA for BC in simulated specimens was 400 CFU/mL; (F) The detection sensitivity of RAA for PF in simulated specimens was 600 CFU/mL; (G) The detection sensitivity of EvaGreen-RAA for BC in simulated specimens was 100 CFU/mL; (H) The detection sensitivity of EvaGreen-RAA for PF in simulated specimens was 200 CFU/mL. Abbreviation: RAA=recombinase aided amplification; BC=Bacillus cereus; PF=Pseudomonas fluorescens.

Figure 3

Dual EvaGreen-RAA assay using melting curves for mixed BC and PF in simulated milk samples with a concentration of 200 CFU/mL for each pathogen. Abbreviation: RAA=recombinase aided amplification; NC=negative control; BC=Bacillus cereus; PF=Pseudomonas fluorescens.